U.S. patent application number 13/607337 was filed with the patent office on 2013-06-13 for formation of bilayers of amphipathic molecules.
This patent application is currently assigned to ISIS INNOVATION LIMITED. The applicant listed for this patent is John Hagen Pryce BAYLEY, Andrew John Heron, Matthew Holden, David Needham. Invention is credited to John Hagen Pryce BAYLEY, Andrew John Heron, Matthew Holden, David Needham.
Application Number | 20130146815 13/607337 |
Document ID | / |
Family ID | 37006184 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130146815 |
Kind Code |
A1 |
BAYLEY; John Hagen Pryce ;
et al. |
June 13, 2013 |
FORMATION OF BILAYERS OF AMPHIPATHIC MOLECULES
Abstract
A method of forming bilayers of amphipathic molecules uses
droplets of aqueous solution in a hydrophobic medium such as oil. A
layer of amphipathic molecules such as a lipid is formed around the
surfaces of the droplets. This may be achieved by providing the
lipid in the oil and leaving the droplets for a time sufficient to
form the layer. The droplets are brought into contact with one
another so that a bilayer of the amphipathic molecules is formed as
an interface between the contacting droplets. The bilayers may be
used for a wide range of studies. The technique has numerous
advantages including providing a long lifetime for the bilayers,
allowing study of small volumes and allowing the construction of
chains and networks of droplets with bilayers in between to study
complex systems.
Inventors: |
BAYLEY; John Hagen Pryce;
(Oxford, GB) ; Holden; Matthew; (Oxford, GB)
; Heron; Andrew John; (Oxford, GB) ; Needham;
David; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYLEY; John Hagen Pryce
Holden; Matthew
Heron; Andrew John
Needham; David |
Oxford
Oxford
Oxford
Durham |
NC |
GB
GB
GB
US |
|
|
Assignee: |
ISIS INNOVATION LIMITED
Oxford
GB
|
Family ID: |
37006184 |
Appl. No.: |
13/607337 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12375103 |
Sep 9, 2009 |
8268627 |
|
|
PCT/GB2007/002856 |
Jul 26, 2007 |
|
|
|
13607337 |
|
|
|
|
Current U.S.
Class: |
252/408.1 |
Current CPC
Class: |
B01L 2400/0427 20130101;
B01L 3/502761 20130101; B01L 3/502792 20130101; B01L 2300/0867
20130101; B01L 2300/0816 20130101; Y10T 436/12 20150115; B01L
2300/161 20130101; G01N 33/5432 20130101; G01N 33/6872 20130101;
G01N 2333/31 20130101 |
Class at
Publication: |
252/408.1 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. FA9550-06-C-0006 awarded by USAF/AFOSR. The government has
certain rights in this invention.
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2006 |
GB |
0614835.7 |
Claims
1-21. (canceled)
22. A droplet of aqueous solution having a volume less than 1000 nL
in a hydrophobic medium, the droplet comprising a layer of
amphipathic molecules around the surface of the aqueous solution,
and containing a membrane protein capable of insertion into a
bilayer of the amphipathic molecules.
23. A droplet according to claim 22, wherein the membrane protein
is a channel or a pore.
24. A droplet according to claim 22, wherein the hydrophobic medium
is an oil.
25. A droplet according to claim 24, wherein the oil is a
hydrocarbon.
26. A droplet according to claim 22, wherein the amphipathic
molecules are lipid molecules.
27. A droplet according to claim 22, wherein the droplet has a
volume greater than 200 nL.
28. A droplet according to claim 22, wherein the droplet has a
volume less than 800 nL.
29. A pair of droplets, a first droplet of the pair of droplets
being a droplet according to claim 22, a second droplet of the pair
of droplets being a droplet of aqueous solution having a volume
less than 1000 nL in the hydrophobic medium, the second droplet
comprising a layer of the amphipathic molecules around the surface
of the aqueous solution, and the first droplet and the second
droplet being in contact with one another so that a bilayer of the
amphipathic molecules is formed as an interface therebetween.
30. A pair of droplets according to claim 29, wherein the bilayer
of the amphipathic molecules has a diameter in the range from 30
.mu.m to 1000 .mu.m.
31. A pair of droplets according to claim 29, wherein the membrane
protein is a channel or a pore.
32. A pair of droplets according to claim 29, wherein the
hydrophobic medium is an oil.
33. A pair of droplets according to claim 32, wherein the oil is a
hydrocarbon.
34. A pair of droplets according to claim 29, wherein the
amphipathic molecules are lipid molecules.
35. A pair of droplets according to claim 29, wherein the droplets
each have a volume greater than 200 nL.
36. A pair of droplets according to claim 29, wherein the droplets
each have a volume less than 800 nL.
37. A pair of droplets according to claim 29, wherein the second
droplet contains a membrane protein capable of insertion into a
bilayer of the amphipathic molecules.
38. A system comprising a bilayer of amphiphilic molecules provided
at the interface between a droplet of aqueous solution having a
volume less than 1000 nL in a hydrophobic medium and a further
aqueous solution, wherein the bilayer contains a membrane
protein.
39. A system according to claim 38, wherein the droplet comprises a
layer of amphipathic molecules around the surface of the aqueous
solution.
40. A droplet according to claim 38, wherein the membrane protein
is a channel or a pore.
41. A droplet according to claim 38, wherein the hydrophobic medium
is an oil.
42. A droplet according to claim 41, wherein the oil is a
hydrocarbon.
43. A droplet according to claim 38, wherein the amphipathic
molecules are lipid molecules.
44. A droplet according to claim 38, wherein the droplet has a
volume greater than 200 nL.
45. A droplet according to claim 38, wherein the droplet has a
volume less than 800 nL.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 12/375,103, filed Sep. 9, 2009, which is the U.S. national
phase of International Application No. PCT/GB2007/002856, filed
Jul. 26, 2007, which designated the U.S. and claims priority to
Great Britain Application No. 0614835.7, filed Jul. 26, 2006, the
entire contents of each of which are hereby incorporated by
reference.
[0003] The present invention relates to the formation of bilayers
of amphipathic molecules such as lipids. Such bilayers are models
of cell membranes and as such may be used to perform a wide range
of studies in biotechnology.
[0004] Lipid bilayers, or more generally bilayers of amphipathic
molecules, are models of cell membranes and serve as excellent
platforms for a range of experimental studies, for example in vitro
investigation of membrane proteins by single-channel recording.
Conditions such as temperature, bilayer composition (surface
charge, cholesterol content etc.) and transmembrane potential can
be adjusted to either mimic biological systems or venture beyond
the physiological range. Most importantly, manipulation of the
solution conditions (pH, salt composition, ionic strength) is
possible on both sides of the membrane. While the in vivo
investigation of membrane proteins in living cells is possible by
the patch clamp method, the conditions that can be used are limited
by the requirement to keep the cells healthy.
[0005] In the conventional technique, planar lipid bilayers are
formed over a plastic aperture which has been pretreated with an
oily mixture. Despite widespread use in academic research, this
conventional technique suffers from a number of limitations, for
example as follows.
[0006] The conventional technique is cumbersome and the bilayers
produced are delicate. The two most common methods of forming
planar bilayers are to paint a lipid/oil mixture across an aperture
and to fold together two monolayers, one from each side of an
aperture. Both techniques require the hands of a skilled scientist.
Small hydrostatic forces due to vibration or flow often rupture
planar bilayers. Even under the best conditions, the lifetime of a
planar bilayer is usually only a few hours. The limited lifetime
effectively limits the range of processes which it is possible to
study.
[0007] Also, since the size and shape of the each bilayer's annulus
region (which governs the membrane's properties) is unique, the
insertion of membrane proteins into planar bilayers is difficult to
standardize.
[0008] Another cause of difficulty when studying some systems is
that the volume of the electrical recording cell on either side of
the planar bilayer is typically greater than 1 mL. Therefore, each
experiment needs large amounts of protein or other reagents, which
may not be easy to produce. After each experiment, both sides of
the electrical recording cell must be thoroughly cleaned.
[0009] Lastly, the conventional technique uses a single planar
bilayer between two chambers. To study systems comprising larger
numbers of membranes, it would in principle be possible to
construct a cell having more chambers and membranes, but the
experimental techniques of forming the bilayers would be very
complicated and in practical terms would make this very labourious
and unattractive to the scientist.
[0010] It would be desirable to develop an alternative method which
alleviates one or more of these limitations.
[0011] According to the present invention, there is provided a
method of forming bilayers of amphipathic molecules,
comprising:
[0012] forming a plurality of droplets of aqueous solution in a
hydrophobic medium with a layer of amphipathic molecules around the
surfaces of the droplets;
[0013] bringing droplets into contact with one another so that a
bilayer of the amphipathic molecules is formed as an interface
between contacting droplets.
[0014] This method of forming bilayers has considerable advantages
and in particular overcomes the limitations of the conventional
technique discussed above.
[0015] The method is straightforward to perform and results in
robust bilayers which can be used in a wide range of studies and
applications in the field of biotechnology. The droplets can be
formed very easily, for example simply by pipetting aqueous
solution into the hydrophobic medium. Similarly, the droplets are
easily moved around to bring them into contact with each other.
Indeed the manner in which droplets are moved is not critical as
the droplets are robust and easily manipulated. Examples of
suitable apparatus for handling the droplets are given below, but
are not limitative.
[0016] The formation of a layer of amphipathic molecules around the
surfaces of the droplets is also straightforward. For example, it
may be achieved simply by providing the amphipathic molecules in
the hydrophobic medium or in the aqueous solution of the droplets,
whereupon the layer can form naturally if the droplets are left for
a sufficient period of time. The amphipathic molecules may also be
dissolved, or suspended as lipid vesicles in the droplets
themselves, from where they again spontaneously form monomolecular
layers at the interface between the droplet and the hydrophobic
medium, that may have an equilibrating concentration of the
amphipathic molecule in the hydrophobic medium.
[0017] The bilayer is formed simply by bringing droplets into
contact with one another. The orientation of the amphipathic
molecules in the layer around the aqueous solution allows the
formation of the bilayer. As the droplets are brought into contact,
after the intervening hydrophobic medium has been displaced the
bilayer forms very quickly as an interface between the contacting
droplets. The bilayer forms a planar surface between the two
droplets which are otherwise generally spherical. This planar
bilayer is the shape with the lowest free surface energy and has a
negative free energy of formation; it is therefore a spontaneous
event. The amphipathic molecules allow two droplets to be brought
into contact without allowing them to coalesce by the formation of
a stable bilayer.
[0018] The droplets may be handled by a variety of techniques. One
particularly advantageous method of moving the droplets is to
dispose an anchor having a hydrophilic outer surface inside a
droplet. Movement of the allows the droplet to be moved, for
example to bring it into contact with another droplet.
[0019] The bilayer can be used to perform experiments involving a
process occurring at or through the bilayer of the amphipathic
molecules. A major class of experiments use a membrane protein
inserted into the bilayer. This may be achieved simply by providing
the membrane protein in the aqueous solution. It has been shown
that after the formation of the bilayer, the membrane protein
naturally inserts into the bilayer in the same manner as with a
bilayer formed by the conventional technique.
[0020] It has been observed that the bilayer behaves functionally
in the same manner as a bilayer formed by the conventional
technique. Therefore the bilayer formed by the present method can
be used to perform the same types of experiments, but providing a
number of advantages which broaden the range of possible
experiments, as discussed further below. Thus the present method
may be applied to a wide range of experiments including
investigation and/or screening of membrane proteins, investigation
and/or screening of analytes which interact with membrane proteins,
and investigation and/or screening of the bilayers. Indeed the
method may be used to study any bilayer phenomena in general,
typically involving a process occurring at or through the
bilayer.
[0021] The lipid bilayer may also be used to study the properties
of the membrane protein inserted therein. For example, the voltage
dependence of the properties of the membrane protein may be
determined Techniques for studying membrane proteins in lipid
bilayers are well known in the art. The function of a channel or
pore may be determined by measuring, for example, an ionic current
flowing across the lipid bilayer through a membrane protein. The
function of a transporter may be determined by measuring the amount
of a molecule translocated across the lipid bilayer, for example by
mass spectrometer or ELISA or by using a substrate which is tagged
fluorescently or radioactively.
[0022] Other examples of experiments which may be performed as
follows.
[0023] The water droplets can be osmotically inflated or deflated
depending on their initial osmolarity. Water can transfer between
the droplets in response to an osmotic gradient through the formed
bilayer at the contact. Also, other molecules that are bilayer
permeable, like drugs, or imageable molecules can be made to move
from one droplet to another through the bilayer contact zone. Thus,
reactants can be separated and allowed to react only when
transported across the bilayer. Here applications may involve
microfluidic systems of droplets that can be brought into contact
and allowed to react to produce new products formed only when the
reactants cross the bilayer contacts between different droplets.
One possibility is that one reactant is bilayer permeable while the
other is not bilayer permeable. In this case the products only
occur in the droplet containing the non-bilayer permeable reactant.
If on the other hand both reactants are bilayer transferable then
the products can be formed in both droplets, or a plurality of
contacting droplets, dependent on the relative permeability of each
reactant across the contact bilayers. These examples do not involve
membrane proteins per se, but just the formed bilayer contacts
through which reactants and products might diffuse.
[0024] Further specific examples of studies to which the present
method may be applied are discussed further below.
[0025] In many studies electrical measurements are taken. This is
straightforward to achieve by bringing electrodes into electrical
contact with the droplets when the droplets are in contact with one
another, for example by insertion of the electrodes into the
droplets or by placing droplets onto static electrodes inserted
into the chamber or in the microfluidic channels
[0026] Bilayers formed by the present method have the advantage of
being robust and having a long lifetime, as compared to the
conventional technique. For example bilayers formed by the
conventional technique require skill to prepare and typically last
a few hours and at most in a very small percentage of cases a
couple of days. In contrast bilayers formed by the present
technique are formed more reliably and last much longer, generally
lasting a number of days. Although a full study of lifetime has not
been performed, a bilayer has been observed to last for a period of
8 days before it was purposely divided by separating the drops.
[0027] It is hypothesised that the reason for the higher lifetime
is that the bilayer formed between two droplets has a lower surface
free energy than a bilayer formed by the conventional technique. In
the latter case, in the annulus region adjacent the periphery of
the aperture, the bilayer divides into two monolayers which extend
on opposite sides of the barrier defining the aperture. It may also
be that stability is conferred by the absence of an annulus that
has to be attached (or adsorbed) onto the material of a septum
defining an aperture. In a similar fashion to the conventional
technique, the bilayer also divides into two monolayers which in
this case simply coat the droplet interfaces, and do not terminate
at a support material (except at the surface of the chamber). The
bilayer is therefore not "stretched" across an aperture, but forms
at the contact zone of the two droplets and no additional spreading
or wetting tensions are induced, just the bilayer tension
determined by the monolayer tension and the free energy of
formation in the hydrophobic medium.
[0028] The higher lifetime allows the study of biological processes
which themselves have a longer lifetime. In this way the present
method opens up new fields of study.
[0029] The formation of the bilayers is also highly reversible and
repeatable. Droplets which have been brought into contact with one
another may be freely separated to divide the bilayer and may be
subsequently brought into contact again to re-create the bilayer.
Such control over the creation, division and re-creation of
bilayers also opens up new fields of study.
[0030] The degree of control makes the formation of the bilayers
easy to standardise. In particular, it is easy to vary the area of
the bilayer of the amphipathic molecules by moving the droplets
when the droplets are in contact with one another. The change in
the area of the bilayers may be observed visually or by capacitance
measurements. It has been demonstrated that it is possible to
change the diameter of the bilayer over the range from 30 .mu.m to
1000 .mu.m, although this is not thought to be the limit.
[0031] In addition, the nature of the hydrophobic medium determines
the degree of spreading of the contacted monolayers and thereby the
contact angle. For example, in experiments it has been observed
that for bilayers of glycerylmonooleate (GMO) formed in decane as
the hydrophobic medium, the contact area is relatively small and
the contact angle is about 3.degree., this being in agreement with
contact angles measured in conventional lipid membrane systems. On
the other hand, if the hydrophobic medium is squalene, a larger
contact area is formed and the contact angle is 25, again in
agreement with measurements on conventional lipid membranes. These
solvent-dependent effects reflect the small free energy of
formation of the GMO:decane system (around -4 mJ/m.sup.2) as
compared to the GMO:squalene system (around -500 mJ/m.sup.2), where
the bilayer thickness concomitantly decreased from 50 .ANG. to 25
.ANG., signifying a depletion of the larger squalene solvent from
the bilayer. This non-linear increase in free energy of formation
departs from simple Lifshitz theory for two infinite slabs of water
acting across the thin oil film, and is more in line with a
"depletion flocculation" effect. Essentially, the larger squalene
solvent molecules are entropically excluded from the GMO bilayer,
and this depletion of solvent exerts a greater osmotic pressure on
the bilayer, thereby raising the free energy of formation by orders
of magnitude in going from decane to squalene, over and above any
Lifshitz effects. Adhesion and the strength and stability of the
contact then are largely dependent on the presence or absence of
solvent in the bilayer.
[0032] Another advantage of the present method is that it allows
the use of a relatively small volume of aqueous solution. In
particular, the volume may be smaller than that present in the
chambers of a cell used in the conventional technique. The droplets
may typically have a volume less than 1000 nL. In general the
droplets may be of any size limited only by the degree of control
of the dispenser of the aqueous solution and the limits of optical
resolution if direct manipulation is desired. Droplets that are not
required to have electrical recording or stimulus from placed
electrodes can be assembled in suspension forming a raft or 3D
aggregate or flocculent of droplets having dimensions of
micrometres to even nanometres that are all in contact with each
other via their intervening bilayers. Using a standard pipette,
experiments have been performed on droplets having volumes in the
range from 200 nL to 800 nL but it is expected that droplets of
smaller volumes could be produced with suitable equipment. For
example, using micro-pipette manipulation to form the droplets from
glass micro-pipettes observed in a relatively powerful microscope,
where droplets of diameter say 30 .mu.m are assembled, the volume
is approximately 14 pL. In suspension, droplet aggregation of
droplets of diameter say 200 nm yields internal volumes of
approximately 4 aL (aL stands for attolitre being 10.sup.-18
L).
[0033] An important advantage of the present invention is that it
is possible to bring more than two droplets into contact with each
other in a chain or network, for example on a flat or dimpled
surface, in a microfluidic channel or, as alluded to above, in
aggregated or flocculated suspension. The simplicity and control
with which the bilayers can be formed simply by moving droplets
around makes it straightforward to build large chains or networks
which would be impractical in a system where bilayers are formed in
apertures in barriers in accordance with the conventional
technique. This opens up the possibility of studying much larger
systems than is practical with the conventional technique, for
example modelling entire systems using multiple droplets. Some
examples are given below, but the range of science which could be
studied is much wider.
[0034] The method can be performed with a wide range of materials,
as follows.
[0035] In general, the amphipathic molecules can be of any type
which form a bilayer in the hydrophobic medium in which the
droplets are positioned. This is dependent on the nature of the
hydrophobic medium and the aqueous solution, but a wide range of
amphipathic molecules are possible. Amphipathic molecules are
molecules which have both hydrophobic and hydrophilic groups. The
layer formed around the droplet is a monolayer of amphipathic
molecules which is formed and maintained naturally by the
interaction of the hydrophobic and hydrophilic groups with the
aqueous solution so that the molecules align on the surface of the
droplet with the hydrophilic groups facing inwards and the
hydrophobic groups facing outwards.
[0036] An important class of amphipathic molecules to which the
present method may be applied is lipid molecules. The lipid
molecules may be any of the major classes of lipid, including fatty
acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids and polyketides. Some
important examples include a phospholipid, a glycolipid or
cholesterol. The lipid molecules may be naturally occurring or
synthetic. Whilst the formation of a bilayer from lipid molecules
has been demonstrated the method is expected to be appropriate for
any amphipathic molecules capable of forming a bilayer.
[0037] The amphipathic molecules need not be all of the same type.
The amphipathic molecules may be mixtures. Another important
example is that the amphipathic molecules in the respective layers
of two droplets brought into contact are of different types so that
the bilayer formed by the two monolayers is asymmetric.
[0038] The aqueous solution may be freely chosen for the
experimental study which is to be performed. The aqueous solution
of each droplet may be the same or different. The nature and
concentration of the solutes can be freely varied to vary the
properties of the solution. One important property is pH and this
can be varied over a wide range. Another important point in
experiments using electrical measurements is to select appropriate
salts to carry the current. Another important property is
osmolarity.
[0039] The hydrophobic medium can also be selected from a wide
range of materials. The material is hydrophobic so that the aqueous
solution forms a droplet rather than mixing with the hydrophobic
medium but otherwise the hydrophobic medium can be freely chosen.
The viscosity of the hydrophobic medium can be selected to affect
the movement of the droplets and the speed of formation of the
layer of amphipathic molecules in the case that they are provided
in the hydrophobic medium.
[0040] The hydrophobic medium may be an oil. Any type of oil is
suitable as long as its surface activity is relatively high, and
that it does not destabilize the formed bilayers. The oil may be a
hydrocarbon which may be branched or unbranched, for example a
hydrocarbon having from 5 to 20 carbon atoms (although hydrocarbons
of lower molecule weight would require control of evaporation).
Suitable examples include alkanes or alkenes, such as hexadecane,
decane, pentane or squalene. Other types of oil are possible. For
example the oil may be a fluorocarbon. This might be useful for the
study of some systems, for example to minimise loss of a particular
membrane protein or analyte from the droplet or to control gas
content such as oxygen.
[0041] As discussed above, in many experimental studies a membrane
protein is provided in one or more of the droplets for insertion
into the bilayer. The present method does not limit the choice of
membrane protein, provided that the aqueous solution is chosen with
appropriate properties for the protein in question. Thus the
membrane protein may be of any type. The use of integral membrane
proteins has been demonstrated, but it is equally expected that
peripheral membrane proteins could be used. The present method
applies to any membrane proteins including the two major classes
that is .beta.-barrels or .alpha.-helical bundles. An important
application is a membrane protein which is a pore or a channel.
Besides a protein pore or channel, further possible membrane
proteins include, but not exclusively, a receptor, a transporter or
a protein which effects cell recognition or a cell-to-cell
interaction.
[0042] To allow better understanding, embodiments of the present
invention will now be described by way of non-limitative example
with reference to the accompanying drawings, in which:
[0043] FIG. 1 is a cross-sectional view of an apparatus for
handling droplets to form a bilayer;
[0044] FIG. 2 is a schematic cross-sectional view of two contacting
droplets with a bilayer formed as an interface therebetween;
[0045] FIG. 3 is a cross-sectional view of an alternative apparatus
for handling droplets to form a bilayer;
[0046] FIGS. 4 to 7 are traces of the ionic current in particular
experiments performed using bilayers formed between two
droplets.
[0047] FIG. 8 is an image of a network of droplets;
[0048] FIG. 9 is a diagram of a network of droplets forming a
"bio-battery";
[0049] FIG. 10 is a trace of current recorded in the network of
FIG. 9;
[0050] FIG. 11 is a diagram of a network of droplets having light
sensing capabilities; and
[0051] FIG. 12 is a trace of current recorded in the network of
FIG. 11.
[0052] An apparatus 1 which is suitable for handling droplets of
aqueous solution to perform the present method is shown in FIG. 1.
The apparatus 1 comprises a container 2 being a 1 mL Perspex bath.
The container 2 contains an oil 3.
[0053] Within the oil 3 are two droplets 4 of aqueous solution. As
the oil 3 is a hydrophobic medium, the aqueous solution of the
droplets 4 does not mix with the oil 3 to any large extent. Some
mutual solubility is expected depending on the solubility limit of
a hydrocarbon in the water and the water in the hydrocarbon. The
droplets 4 are formed into the oil 3 simply by dispensing the
aqueous solution, for example from a conventional pipette or indeed
any suitable dispenser. The dispenser is preferably of a type which
allows the volume of the droplets 4 to be controlled. Experiments
have been performed using droplets 4 of volume in the range from
200 nL to 800 nL but this is not limitative. One of the advantages
of the method is the ability to use droplets 4 of small volume and
it is expected that volumes less than 200 nL could be used.
Micro-pipette manipulation has been used to make droplets 4 of only
tens of micrometres and it is anticipated that emulsification
techniques can produce suspensions of bilayer contacting droplets
that have diameters of order 100 nm.
[0054] On the base of the container 2 within the oil 3, the
apparatus 1 is provided with three supports 5 having a hydrophobic
outer surface. The supports 5 are in this example simply 10 .mu.L
disposable pipette tips mounted on the container 1. Thus the
supports 5 are annular. The droplets 4 may be disposed on the
supports 5 by dispensing them in this location. By way of example,
FIG. 1 shows a droplet 4-A supported on one of the supports 5. The
hydrophobic nature of the outer surface of the support 5 which
arises due to the support 5 being made of plastic prevents the
droplet 4-A supported thereon from flowing down over the support 5.
As an alternative there could be used a support in the form of a
dimpled surface.
[0055] Each support 5 is provided with an anchor 6 formed by a
hydrogel droplet 7 held on the end of an electrode 8 formed by a
100 .mu.m diameter rod coated with Ag/AgCl which protrudes 0.5 mm
through the aperture in the centre of the support 5 so that the
hydrogel droplet 7 is disposed inside the droplet 4-A of aqueous
solution. In particular the hydrogel is 5% (w/v) agarose in buffer
solution. Due to hydrogel having a very high water content, the
outer surface of the hydrogel droplet 7 is hydrophilic. As a result
the hydrogel droplet 7 anchors the droplet 4-A supported on the
support 5 because of the attraction to the aqueous solution of the
droplet 4-A. In this way, the anchor 6 assists in holding the
droplet 4-A on the support 5.
[0056] Further droplets 4, for example the droplet 4-B shown in
FIG. 1, may be moved around within the oil 3 using a
micromanipulator 9 which is shown schematically in FIG. 1 and may
be of a conventional type. The droplet 4-B is held by an anchor 10
connected to the micromanipulator 9. The anchor 10 comprises a
section of Ag wire which has been partially melted at the end to
form a 200 .mu.m diameter ball. This was first treated with NaClO
to create an Ag/AgCl electrode 12 and then coated with a layer of
hydrogel to form a hydrogel droplet 11. In particular the hydrogel
is 5% (w/v) agarose in buffer solution and has a thickness of order
200 .mu.m. Due to hydrogel having a very high water content, the
outer surface of the hydrogel droplet 11 is hydrophilic. As a
result the hydrogel droplet 11 anchors the droplet 4-B on the
anchor 10 because of the attraction to the aqueous solution of the
droplet 4-B. Thus the droplet 4-B may be moved around by
controlling the micromanipulator 9 to move the anchor 10.
[0057] After the droplets 4 of aqueous solution are formed in the
oil 3, a layer of amphipathic molecules, such as lipid molecules,
are formed around the surfaces of the droplets 4. There are two
options for achieving this.
[0058] The first option is to provide the amphipathic molecules in
the oil 3. This may be done before dispensing the droplets 4 into
the oil 3, that is by providing the oil 3 as a solution of the
amphipathic molecules in the oil 3. Alternatively, the amphipathic
molecules could be provided after dispensing the droplets 4 into
the oil 3 but in that case it is harder to mix the amphipathic
molecules with the oil 3. The droplets 4 themselves may contain the
surfactant or the lipid in solution, micellar suspension or as
lipid vesicles or liposomes.
[0059] Subsequently, after the amphipathic molecules have been
provided in the oil 3 and after dispensing the droplets 4 into the
oil 3, a layer of amphipathic molecules forms around the outer
surface of the droplets 4 spontaneously. This can be achieved
simply by leaving the droplets 4 in the oil 3 for a sufficient
period of time. It is not necessary to take any special measures to
encourage formation of the layer, but measures such as agitation
might speed up the process.
[0060] The second option is to provide the amphipathic molecules in
the aqueous solution dispensed into the oil 3 to form the droplets
4. For example, the amphipathic molecules may be provided as
vesicles suspended in the aqueous solution. Subsequently, the layer
of amphipathic molecules forms around the outer surface of the
droplets 4 spontaneously. This can be achieved simply by leaving
the droplets 4 in the oil 3 for a sufficient period of time. It is
not necessary to take any special measures to encourage formation
of the layer, but measures such as agitation might speed up the
process.
[0061] The period of time required to form the layer of amphipathic
molecules depends on the nature of the oil 3, the amphipathic
molecules and the aqueous solution, but is typically of the order
of tens of minutes. The required time is easily determined
experimentally for any given material system. That is to say,
trials can be performed in which droplets 4 are formed and left for
different periods of time before the droplets 4 are brought
together to form a bilayer as described below. Trials where the
period of time is too short will not result in a stable bilayer so
instead the droplets 4 merge together to form a larger droplet. The
period of time in respect of the trials where a stable bilayer does
form is the appropriate period for the material system in
question.
[0062] After the layer of amphipathic molecules has formed around
the droplets 4, the droplets 4 are brought into contact with one
another. This is achieved in the apparatus of FIG. 1 by moving the
droplet 4-B using the micromanipulator 9 until it is in contact
with the static droplet 4-A supported on the support 5.
[0063] When the droplets 4 are brought into contact a bilayer of
the amphipathic molecules forms as the interface between the
droplets 4. This is illustrated in FIG. 2 which shows two droplets
4 of aqueous solution in the oil 3. Each droplet 4 is surrounded by
a layer of amphipathic molecules 13 which are shown schematically
(and not to scale) oriented with their hydrophobic tails 13a facing
outwards and their hydrophilic heads 13b facing inwards. Where the
droplets 4 come into contact, the layers of amphipathic molecules
13 of each droplet 4 form a bilayer 14. As it is the shape of
lowest free surface energy, the bilayer 14 is planar, at least as
compared to the monolayer of amphipathic molecules 13 around the
remainder of the droplets 4 (the bilayer 14 may have some small
degree of curvature).
[0064] The formation of the bilayer occurs spontaneously when the
droplets 4 come into contact and may be observed visually through a
microscope. As the droplets 4 come into contact there is a short
delay where the droplet 4 deform and then spontaneously the bilayer
14 forms with a planar shape as shown in FIG. 2. The delay is the
time taken for the oil 3 between the droplets 4 to be displaced out
of the interface.
[0065] The formation of the bilayer 14 can also be observed by
electrical measurements, in particular of the capacitance between
the droplets 4. To measure this and perform other electrical
measurements, the apparatus 1 further includes a circuit 15 of the
same type as used in known apparatuses for studying bilayers using
the conventional method described above using an aperture in a
barrier. The circuit 15 is connected to the electrodes 8 and 12. An
electrical contact with the aqueous solution of the droplets 4 is
made due to the conductive nature of the hydrogel droplets 7 and
11. When a bilayer 14 forms, the capacitance measured by the
circuit 15 increases in the same manner as with a bilayer formed by
the conventional method.
[0066] A particular advantage of the present method is that the
area of the bilayer 14 can be varied by moving the contacting
droplets away and towards each other. In the apparatus 1 of FIG. 1,
this is achieved by movement of the droplet 4-B by the
micromanipulator 9. The changing area of the bilayer 14 can be
observed both visually and from the capacitance measurements. In
experiments the average diameter of the bilayer 14 has been changed
in the range from 30 .mu.m to 1000 .mu.m although this is not
limitative. Capacitance measurement also allows for precise control
of the area of the bilayer 14 which can provide the advantage that
a given experiment can be standardised by using a bilayer 14 of
standard area. Another advantage of varying the area is that
insertion of a membrane protein can be encouraged by initially
forming a large bilayer 14 and after insertion the area of the
bilayer 14 can be reduced. It has been observed that during
reduction of the area of the bilayer 14, the membrane protein
remains inserted, as observed by measurements, until just before
the bilayer 14 separates. Such reduction in the area of the bilayer
14 can also reduce noise in electrical measurements.
[0067] It has also been observed that the bilayer 14 can be
repeatedly and reliably separated by separating the droplets 4 and
re-created by bringing the droplets 4 back into contact. This is
advantageous as it allows complex experiments to be performed.
[0068] The apparatus 1 is convenient but in general droplets 4 of
aqueous solution can be brought together to form a bilayer 14 in a
wide range of apparatuses. The droplets 4 themselves are
sufficiently robust to be manipulated in a number of different
ways, for example by being physically pushed or placed, instead of
anchoring them to an anchor. Similarly, the formation of the
bilayer 14 is robust and repeatable not dependent on how the
droplets 4 are manipulated. Thus a variety of different apparatuses
can be used depending on the application. The manipulation
technique may be very simple. For example, the droplets 4 may be
moved simply by pushing them using a simple probe such as a glass
or plastic rod. Alternatively, more complex manipulation techniques
may be applied. For example the droplets may be moved using
micro-fluidic apparatus. There is extensive discussion in the
literature of micro-fluidics being used to move droplets of aqueous
solution in oil and such techniques may be advantageously applied
to the present invention, for example to facilitate high
through-put screening.
[0069] A simple, alternative apparatus 16 which has been used is
shown in FIG. 3. In this case, the base of the container 2, which
in this case is made of teflon, has electrodes 17 protruding
through the base. The droplets 4 are supported on the base
enveloping the electrodes 17. The droplets 4 are positioned simply
by dispensing them in the oil 3 and subsequently manipulated by
pushing them around. For example, droplets 4 may be dispensed onto
the electrodes 17 or may be dispensed to a different area of the
base and pushed onto the electrodes 17. Although the apparatus 1 of
FIG. 1 is more convenient to use, the apparatus 16 of FIG. 3 does
allow bilayers 14 to be formed and monitored, thus demonstrating
the robustness of the technique.
[0070] Conversely it is anticipated that other more complicated
apparatuses could be used, depending on the application. For
example for use in screening, an array of droplets 4 might be
supported and one or an array of further droplets 4 might be moved
relative thereto. Another possible way to move the droplets 4 would
be to use micro-fluidics equipment or electrical patterned
equipment.
[0071] As already discussed, the bilayers 14 formed by the present
method may be used to perform a wide range of experiments involving
a process occurring at or through the bilayer of the amphipathic
molecules. Some examples of actual experiments which have been
performed to demonstrate the efficacy of the present method are
given below.
[0072] For example, having established that droplets 4 adhere and
form a bilayer 14 at the contact zone, it is of interest to measure
the water permeability of the bilayer 14 such as in the GMO/solvent
bilayer system. This easily accomplished by simply forming two
droplets 4 each droplet 4 having different osmotic pressure. In the
experiment carried out, we chose to assemble one droplet 4 of
solution of 500 mOsm glucose and the other droplet 4 of pure
deionized water. The two micro-pipettes that form the droplets 4
are filled each with the different solution, and using the pressure
control, positive pressure is applied to first one and then the
other to generate the two droplets 4. Forming the droplets 4 in
GMO-squalene ensures that water is not rapidly lost to the
surrounding hydrocarbon phase and that changes in volume of the
droplets 4 represents the passage of water from the water droplet 4
to the glucose solution droplet 4 down the osmotic gradient,
through the bilayer 14 formed at the contact. The experiment is
then to simply assemble the droplets 4 in to adherent contact and
record on video their progress and volume changes (calculated from
the diameters) that occur due to water transport. The glucose
solution droplet 4 takes up the water from the pure water droplet 4
and grows as the water droplet 4 shrinks Data is then plotted as
droplet volume with time.
[0073] In many types of experiment, the physical process is
monitored by taking electrical measurements, eg of capacitance,
current or voltage, using the circuit 15 in a similar manner to
known techniques. To this end, the circuit 15 may, by way of
example, comprise a patch-clamp amplifier (Axopatch 200B; Axon
Instruments), filtered with a low-pass Bessel filter (80 dB/decade)
with a corner frequency of 2 kHz and then digitized with a DigiData
1320 A/D converter (Axon Instruments) at a sampling frequency of 5
kHz. The container 1 and amplifying headstage of the circuit 15 may
be enclosed in a metal box to serve as a Faraday cage.
[0074] By way of example, the electrical measurement may measure
the current representative of the passage of ions through a
membrane protein which is an ion channel. However, it is not
essential to use electrical measurements, as any measurement
characterising the process in question may be used. One alternative
to electrical measurement is optical measurement, for example of a
fluorescent molecule which is transported across the bilayer 14 or
which responds to another molecule transported across the bilayer
14 or to the potential across the bilayer 14.
[0075] Many experimental techniques involve the insertion of a
membrane protein into the bilayer 14, the process under study
relating to the function of the membrane protein. In this case, the
membrane protein may be used by providing it in the aqueous
solution of one or more of the droplets 4. The membrane protein
then inserts spontaneously into the bilayer 14. This can be
achieved simply by leaving the bilayer 14 until insertion occurs
without taking any special measures.
[0076] Above, only a single bilayer 14 between two droplets 4 has
been considered. However, it is a particular advantage of the
present method that more than two droplets 4 may be brought into
contact to form plural bilayers 14 between the contacting droplets
4. The droplets 4 may be arranged in a chain or network. For
example, in the apparatus 1 of FIG. 1, three droplets 4 may be
arranged on the three supports 5 with the droplets 4 contacting
each other in a chain, or alternatively droplets 4 on the three
supports 5 may be interconnected by other droplets 4 to form a
longer chain. Such chains of droplets may be branched by connecting
further droplets 4 to the chain. Branches of droplets 4 may be
connected to other droplets 4 to form a network. It would be
straightforward to expand the apparatus 1 of FIG. 1 or indeed any
apparatus to accommodate large numbers of droplets 4 and more
electrodes. Similarly in microfluidic channels two or more droplets
4 can be conjoined to make a chain, or positioned in a network
pattern of channels.
[0077] As the present method of forming bilayers 14 between
droplets 4 is reliable and highly repeatable, this opens up the
possibility of forming and studying complex systems of droplets 4
with bilayers 14 in between which would in practical terms be
prohibitively difficult to do with the conventional method of
forming bilayers across an aperture in a barrier. Furthermore,
multiple interactions could be studied in sequence or in parallel.
The possibilities are wide-ranging and exciting, and include
modelling of tissues such as heart tissue or any monolayer of
epithelia, such as might exist in the gut, or retina, or ear where
gap junctions provide cell to cell communication and redistribution
of ions. Also, endothelial monolayers could be modelled.
[0078] Another possibility is for the aqueous solution of the
droplets 4 to include solutes which cause transport through the
membrane proteins, being either primary or secondary transport. In
this way, the system may be self-powered. Alternatively, the system
may be externally powered, for example electrically by the circuit
15 or optically with light activated systems.
[0079] To illustrate the efficacy of the present method there will
now be described some experiments which have been performed using
the apparatus 1 of FIG. 1.
[0080] The first experiment demonstrates that ion-conducting
membrane proteins insert in the bilayer 14 allowing measurements of
ionic current to be performed when a potential is applied across
the bilayer 14.
[0081] In particular, two 200 nL droplets 4 were arranged on the
support 5 and on the anchor 10, in both which the electrodes 8 and
12 were made of Ag/AgCl. The oil 3 was hexadecane (Sigma) and the
lipid 1,2-diphytanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
(DPhPC, Avanti) was dissolved therein to form a 10 mM solution.
[0082] One droplet 4 contained an aqueous solution of 10 pg/ml
wild-type (WT) staphylococcal .alpha.-hemolysin (.alpha.HL)
heptamer in 10 mM MOPS, 1M KCl, pH 7.0, while the other contained
an aqueous solution of 10 .mu.M .gamma.-cyclodextrin (.gamma.CD,
Sigma) also in 10 mM MOPS, 1M KCl, pH 7.0. The .gamma.CD binds to
WT .alpha.HL and serves as a reversible blocker, which acts as a
diagnostic to show that increases in current during an applied
potential are due to pore insertion rather than current leakage
through the droplet/droplet interface.
[0083] With this material system, it was found that the time
required for the layer of lipid to form around the droplets 4 was
about 30 minutes, i.e. even though lipid is expected to be adsorbed
relatively quickly upon formation of the water-oil interface, it
takes about 30 minutes to establish the relatively highly dense
monolayer required for bilayer formation, a density on the order of
60 .ANG..sup.2 per molecule for this particular lipid. If the
droplets 4 were kept separated for this time and then brought into
contact, a bilayer 14 formed but if the droplets were left for a
lesser time, the bilayer 14 was not stable and the droplets 4
merged in less than one minute after being brought into
contact.
[0084] After formation of the bilayer 14, .alpha.HL pores inserted
therein. The time required for bilayer formation (monitored by
capacitance measurements) was between five and ten minutes. The
ionic current was measured, an example being shown in the trace of
FIG. 4. The conductance of a single .alpha.HL pore was 798.+-.70 pS
(n=6) at -50 mV, similar to a previously reported value of 775 pS
(5 mM HEPES, 1M KCl, pH 7.4). The current was transiently
attenuated by .gamma.CD binding. The residence time (t.sub.off) for
.gamma.CD bound to the .alpha.HL pore was 495.+-.51 ms (n=4) and
was similar to the residence time (t.sub.off=421.+-.22 ms, n=4)
found in control experiments using a folded planar bilayer under
the same solution conditions.
[0085] The form of the signal and the measurements demonstrate that
the function of the membrane protein in the bilayer 14 is
indistinguishable from those derived from a planar bilayer formed
by the conventional method. In contrast the bilayer 14 had a much
greater lifetime, routinely lasting several days.
[0086] The spacing between the droplets 4 was controlled by
movement of the micromanipulator 9. After formation of the bilayer
14, moving the droplets 4 closer together causes the area of the
bilayer 14 to expand, while moving the droplets 4 apart causes the
area of the bilayer 14 to shrink. Although the bilayer 14 was thin
enough (<5 nm) to accommodate protein insertion, it was so
stable that single channel recording was possible even as the
droplets 4 were pushed together or pulled apart, this probably
being a consequence of the relatively large negative free energy of
formation for this structure in this solvent-lipid system.
[0087] It was also demonstrated that the droplets 4 could be
connected and disconnected repeatedly, allowing examination of a
protein-containing droplet 4 many times before loss of protein
activity. As an example of this, a droplet 4-A containing WT
.alpha.HL was screened against an array of three analyte-containing
droplets 4-B, of which: a first droplet 4 contained only buffer (10
mM MOPS, 1M KCl, pH 7.0), the second droplet 4 contained 50 .mu.M
.gamma.CD in buffer, and the third droplet 4 contained 50 .mu.M
TRIMEB (a permethylated .beta.CD) in buffer. The droplet 4-A
containing WT .alpha.HL was placed on the movable cis electrode 12
supported by the micromanipulator 9 while the trans electrode 8 was
common to the three analyte droplets 4-B, each of which was placed
on one of the supports 5.
[0088] The droplet 4-A containing WT .alpha.HL was connected to the
first droplet 4-B and an electrical recording was taken. Pore
insertion was manifested as stepwise increases in ionic current.
After recording, the droplet 4-A containing WT .alpha.HL was
disconnected from the first droplet 4-B and moved to the second
droplet 4-B, and another recording performed and so on. The
transient binding of .gamma.CD (second droplet 4-B) to an .alpha.HL
pore blocked around 60% of the ionic current, while the binding of
TRIMEB (third droplet 4-B) blocked .alpha.HL almost completely.
These results show repeated use of the droplet 4-A containing WT
.alpha.HL. Furthermore after recording from the third droplet 4-B,
the droplet 4-A containing WT .alpha.HL was reconnected to the
first droplet 4-B and a final recording was taken. The .alpha.HL
behaviour was identical to the behaviour shown in the first scan,
demonstrating that the WT .alpha.HL sample had not been
contaminated by either of the blocking analytes.
[0089] Typically, WT .alpha.HL monomer is generated by coupled in
vitro transcription and translation (IVTT) by using an E. coli S30
extract. The protein is then oligomerized on red blood cell
membranes and purified by gel electrophoresis. Since droplets under
other circumstances can serve as nanoreactors, this idea was used
to run the IVTT reaction directly inside a droplet 4.
[0090] In particular, using the apparatus 1 of FIG. 1 two droplets
4 were brought together. The first droplet 4 contained an IVTT mix
expressing WT .alpha.HL, while the second droplet 4 contained 10
.mu.M .gamma.CD in 2.5 mM MOPS, 250 mM KCl, pH 7.0. Although the
IVTT reaction is usually performed at KCl concentrations of less
than 50 mM (Promega, TB129), 250 mM KCl was added to the IVTT mix
to aid in the ionic current recordings.
[0091] In the experiments two .alpha.HL pores inserted into the
bilayer. This was observed by the characteristic increases in the
ionic current at -50 mV and by .gamma.CD binding as shown in the
example trace of FIG. 5 in which current levels 0, 1 and 2 show,
respectively, both pores open: one pore open and one pore partially
blocked by .gamma.CD; and both pores partially blocked by
.gamma.CD. There is at least a two minute lag time between the
mixing of IVTT components and completion of the .alpha.HL
polypeptide chains. Since the droplets were placed into the oil 3
within one minute of adding the final IVTT component, the proteins
must have been produced within the first droplet 4.
[0092] From an electrical perspective, the bilayer 14 and a pore
inserted therein are components of a circuit, in which the bilayer
14 is a capacitor, the pore is a high-resistance conductor and the
ionic solution is a wire that connects the two elements to a
voltage supply. This can be used to establish the basis for more
complex bio-nano-circuitry by assembling droplets 4. An example of
this is now described.
[0093] Three droplets 4 were brought together in a chain, with the
electrodes in the terminal droplets. The first droplet 4 contained
10 mM MOPS, 1M KCl, pH 7.0. the second (centre) droplet 4 contained
WT .alpha.HL heptamer in MOPS buffer. The third droplet 4 contained
10 .mu.M .gamma.CD in MOPS buffer. The first and third droplets 4-A
were arranged on the cis and trans electrodes respectively on a
support 5, while the second droplet 4-A was held with the
micromanipulator 9.
[0094] After formation of two bilayers 14 one between the first and
second droplets 4 and the other between the second and third
droplets 4, the ionic current was monitored and was observed only
after at least one protein had inserted into each bilayer 14.
However, since the formation of each bilayer 14 occurred
independently, it is likely that the bilayer 14 which formed first
began to incorporate pores before the second bilayer 14 formed. A
example current trace, which shows the binding of .gamma.CD to the
pores at the second bilayer 14, is shown in FIG. 6
[0095] The electrical properties of .alpha.HL pores and planar
bilayers have been extensively studied and are straightforward to
simulate in a computer model. In a single bilayer system, the
.gamma.CD binding caused a sharp attenuation of current. However,
when the .gamma.CD bound to an .alpha.HL pore at the interface of
the double bilayer system, both the binding and release were
followed by slow current shifts to the expected levels (note the
curvature in the current trace of FIG. 6 immediately following
binding and release of .gamma.CD). A simulation of this
bio-nano-circuit also predicted this type of binding behaviour.
From the model, we attribute the slow current changes as follows.
The resistance of the pore at the second bilayer 14 increased when
the .gamma.CD bound, which lowered the potential across the first
bilayer 14. This in turn caused the first bilayer 14 (capacitor) to
release some of its stored charge. Upon .gamma.CD release, this
process was reversed. Our results suggest that the behaviour of
chains and networks of droplets 4 is governed by basic electrical
principles and are straightforward to predict. Therefore, one might
engineer circuits to have specific functions, such as feedback
loops.
[0096] The ability to interconnect stable, compartmentalized and
communicating nanolitre volumes, or even picolitre volumes, using
protein gateways forms the basis for the creation of a rudimentary
artificial cell. Just as living cells carry out the functions of
life in separate compartments, small networks of droplets 4 might
be designed to mimic these processes. Also, just as single cells
carry out functions and conjoined cells create tissue, this concept
could be extended to include epithelial and endothelial monolayers
of droplet cells, where even the size scale of 10 .mu.m per cell is
matched, allowing diffusive distances to be equivalent. This is
important because times for diffusive processes are governed by a
square relationship to distance, and so droplets 4 of diameter 100
.mu.m would have diffusion times that are 100 times longer than
droplets 4 of diameter 10 .mu.m.
[0097] The .alpha.HL protein is an excellent starting point, since
the properties of the pore, such as conductivity, ion selectivity,
gating and blocking, and selective transport of small molecules can
be tailored through genetic engineering. Further, .alpha.HL adopts
a known orientation in the membrane, meaning that the direction of
a chemical gradient can be controlled through the arrangement of
the droplets 4 in a network.
[0098] An ionic gradient might be combined with ion selective pores
to generate a transmembrane potential and current across one
droplet interface, which in turn could be used to power processes
occurring at a bilayer 14 farther along a chain of droplets 4.
[0099] The latter concept was demonstrated using a chain of three
droplets. The first droplet 4 contained N123R .alpha.HL
homoheptamer (in 10 mM HEPES, 100 mM NaCl, pH 7.5) which is anion
selective. The second (centre) droplet 4 contained 10 mM HEPES, 1M
NaCl, pH 7.5. The third droplet 4 contained 10 .mu.M .beta.CD and
M113F/K147N .alpha.HL homoheptamer in 10 mM HEPES, 1M NaCl, pH 7.5
buffer. The first and third droplets 4 were connected electrically
to the electrodes.
[0100] After formation of two bilayers 14 one between the first and
second droplets 4 and the other between the second and third
droplets 4, the ionic current was monitored. The ionic gradient
across the first bilayer 14 generated a potential, while the
insertion of pores at both bilayers 14 allowed this potential to be
dissipated as ionic current. The selectivity of the N123R pores
preferentially allowed the flow of Cl- ions from the first droplet
4 to the second droplet 4, which resulted in a positive potential
at first bilayer 14. It should be noted that the circuit 15 was not
used to apply a potential, rather it was only used to record the
current. As shown in the example trace of FIG. 7, the power
supplied by the battery effectively formed by the first and second
droplets 4 enabled the observation of blocking events at the second
bilayer 14, where the M113F/K147N pores reversibly bound the
molecular adapter .beta.CD
[0101] The droplets 4 may be used to create simulated biosystems.
It has been shown that a bilayer 14 spontaneously forms and lasts
several days, which might allow slow processes, such as a complete
metabolic cycle, to be studied. Proteins can be produced in situ
(by IVTT) and studied by single-channel recording in the same
droplet 4. The ability to disconnect and reconnect droplets 4
suggests that this approach might be a powerful tool in
high-throughput screening and combinatorial chemistry applications.
Further, the creation of complex networks is readily accomplished
by arranging droplets 4 in a pattern, the geometry of which need
not be restricted to two dimensions. For example the droplets 4
could be layered in say a hexagonal ABAB or ABCABC "crystal"
pattern. The chain of three droplets demonstrates the feasibility
of connecting nano-compartments with functional gateways and serves
as a possible starting point for mimicking the biological
hierarchy.
[0102] A network of droplets has been demonstrated as follows.
Droplets 4 were created using a straight 20 cm section of 1.59 mm
I.D. tubing that was filled with the oil/lipid mixture and closed
at one end. With the open end up, droplets 4 were pipetted into the
top of the tube just under the surface of the oil and allowed to
fall nearly to the bottom of the tube. The tube was then inverted,
causing the droplet 4 to descend towards the open end. Just before
the droplet 4 reached the opening, the tube was brought into
contact with the surface of oil 3 in a container 2 which allowed
the droplet 4 to land on the bottom of the container 2.
[0103] To form the network the base of the container had a Perspex
surface with a square array of micromachined dimples (a miniature
"egg-crate"), with a diameter of 1 mm and with a centre to centre
spacing of 700 .mu.m each dimple acting as a support for a droplet
4. Electrodes were threaded through 200 .mu.m diameter holes that
were drilled through the bottom of the dimples. The underside of
the cell was sealed with UV curable glue to ensure that the oil 3
did not leak around the electrodes. All electrodes were soldered to
a common wire which was connected to the amplified (as opposed to
grounded) end of a patch clamp headstage.
[0104] For visibility, each droplet 4 contained either
tetramethylrhodamine (pink) or Alexa 488 linked to a dextran
polymer (yellow), in 10 mM MOPS, 1M KCl, pH 7.0. When a droplet 4
was added, it formed a bilayer 4 with its neighbouring droplets 4
as the interstitial oil 3 was displaced. The resultant network of
droplets is shown in FIG. 8.
[0105] The interface between the droplets 4 was stable to
mechanical perturbation. Indeed, it was possible to puncture into a
droplet 4 and then extract it by using an agarose gel-coated
Ag/AgCl electrode controlled by micromanipulator. Further, it was
possible to replace the missing droplet 4 by stabilizing and
dropping a further droplet 4 into the empty position. This droplet
spontaneously integrated into the network. Thus, component droplets
of the network could be extracted and exchanged without
compromising the integrity of the surrounding system.
[0106] Living tissue is differentiated into regions of specific
function, which are in turn sub-differentiated into various cells.
One can envision using the networks of droplets 4 in a similar
fashion, with clusters of droplets 4 dedicated to certain
functions. The interconnection of these clusters might eventually
lead to a rudimentary artificial tissue system mimicing processes
in living cells.
[0107] Membrane proteins may be incorporated, for example pores.
The .alpha.HL protein pore is an excellent starting point, where
the pore adopts a known orientation in the membrane, the position
of protein domains (namely, the cap and stem of the pore) can be
easily controlled by the arrangement of droplets 4. Further, the
properties of the pore, such as unitary conductance, ion
selectivity, rectification, gating, interactions with blockers and
selective transport of small molecules can be tailored through
genetic engineering to provide specific functions in a network.
[0108] Of immediate interest are electrically propagating systems,
such as the heart. Droplets containing ion gradients, gap junctions
and other proteins could be arranged in the correct order to
simulate and study the mechanism of the cardiac impulse
propagation. Since the droplets 4 can be disconnected and
interchanged, libraries of mutant proteins could be screened using
a functional network to study disease related protein
irregularities. For example, an ionic gradient might be combined
with an ion selective pore to generate a transmembrane potential
across one bilayer 14, which in turn could be used to power
processes occurring at a bilayer 14 farther along a chain of
droplets 4 which has been demonstrated as discussed above.
[0109] The properties of a network can be modified by changing its
geometry. For example, a branched "bio-battery" was constructed as
shown in FIG. 9 from six 200 nl droplets 4 using the same ionic
gradient as described above. Three droplets 4a contained N123R
.alpha.HL homoheptamer (in 10 mM HEPES, 100 Mm NaCl, pH 7.5) and
were situated on the termini of a common branched electrode. These
interfaced with three sides of an empty droplet 4b containing 10 mM
HEPES, 1M NaCl, pH 7.5, the remaining side of which droplet 4b was
linked to a short chain of 10 mM HEPES, 1M NaCl, pH 7.5 droplets 4c
containing 17 ng/ml WT .alpha.HL heptamer. The opposing electrode
was plugged into the terminal .alpha.HL droplet 4c. When all
droplets 4a containing N123R .alpha.HL were connected with the
terminal .alpha.HL droplet 4c, a high current (around -390 pA) was
recorded as shown in FIG. 10. As indicated by the arrow 1, one
.alpha.HL droplet 4c was then removed from the network, which
caused the current to drop to around -61 pA. As indicated by the
arrow 2, removal of the second .alpha.HL droplet 4a caused a
further decreased in current to around -21 pA. As indicated by the
arrow 3, removal of the droplet 4b stopped the current.
[0110] Nature's ability to receive and transmit information
gathered from stimuli is enabled by differentiated cells working
collectively. The retina, for example, senses light using rod and
cone cells, which initiate a cascade of processes that transmit
information down the optic nerve for interpretation by the brain.
Droplet "cells" that detect light could be connected to droplet
"cells" that conduct the current, much as in the retinal and
retinal nerves. In fact other "senses" like taste and smell that
are driven by receptor binding and channel conduction even from the
hydrocarbon phase binding to receptors that are positioned in the
monolayers of the droplets 4 could communicate between and within
the droplet networks. Here, receptors that are probably not natural
with a hydrophobic portion bounded by hydrophilic termini, would
likely not partition or orient in a functional form in the droplet
monolayer. But one can envision a hydrophobic domain bounded by
just one hydrophilic domain that anchors the "receptor" in the
monolayer facing in towards the water with the hydrophobic receptor
in the oil phase. If such a molecule could be designed to respond
to hydrophobic soluble molecules binding (most fragrances and
active drugs are relatively hydrophobic), say by a conformational
change that initiated a detectable event inside the droplet
interface, then sensing of analytes ion hydrophobic solution might
be possible.
[0111] In a rudimentary mimic of the retina and optic nerve a
light-sensing network was constructed based on the light-driven
proton pump, bacteriorhodopsin (BR) as shown in FIG. 11. Three
droplets 4d were placed on the termini of a common electrode and
contained 10 mM HEPES, 100 mM NaCl, pH 7.5, 0.001% dodecylmaltoside
(DDM) and 18 .mu.M BR. A central droplet 4e contained 10 mM HEPES,
100 mM NaCl, pH 7.5, while the final outer droplet 4f contained 10
mM HEPES, 100 mM NaCl, pH 7.5 with 17 ng/ml WT .alpha.HL heptamer
and had an opposing electrode plugged into it. A 1 mW green (532
nm) pen laser was used to illuminate the network. As shown in FIG.
12, when the laser was switched on, a sharp spike in current was
visible, which quickly decayed to around 5 pA after 5 seconds.
Switching the laser off caused the current to briefly dip to a
negative value before returning to zero. Similar observations of BR
behaviour have been observed using analogous systems. Three cycles
of 5 seconds on and 5 seconds off were performed, followed by a
rapid sequence of 16 laser pulses. Each BR transports one proton
across the membrane per photon of light absorbed. Therefore, a 5 pA
current suggests that tens of thousands of molecules must be
functioning in the membranes 14. While such large currents might
have been difficult to obtain with a single bilayer, the network of
droplets 4 amplifies the light collecting ability of the system. As
a control, the droplets 4 were replaced with droplets 4 containing
only buffer, and the experiment was repeated. Although the
electrode surfaces were exposed to the laser during illumination,
no current from a photoelectric effect was observed.
[0112] In the above described experiments, the following techniques
were applied. Wild-type (WT), the M123R and M113F/K147N .alpha.HL
heptamers were prepared by in vitro transcription and translation
(IVTT), followed by oligomerization on red blood cell membranes.
After purification by SDS-PAGE, the heptamer band was cut from the
gel and the protein was extracted. Typically, .alpha.HL samples
were diluted between 100 to 10,000 times in the buffer that was
used to form the droplets 4. After dilution, any detergent
remaining from the gel purification did not affect the stability of
the bilayer 14.
[0113] Bacteriorhodopsin (BR) from Halobacterium salinarum was
purchased from Sigma. Without purification, 1 mg of BR was
solubilized by sonication for 30 minutes in 40 .mu.l of a one to
one mixture of buffer (10 mM HEPES, 100 mM NaCl, pH 7.5) and 0.01%
dodecylmaltoside (DDM) in water, which yielded a dark purple
suspension. When preparing BR droplets, the stock suspension of BR
was diluted by a factor of 10 in 10 mM HEPES, 100 mM NaCl, pH
7.5.
* * * * *